Redirected T cell therapies Flashcards
1
Q
How did cellular immunotherapy start?
A
- Cellular immunotherapy really started as HSC transplantation for the treatment of pts with blood cancers
o Patient with leukaemia given chemo with aim of eliminating any residual leukaemia – also eradicates residual haematopoiesis
o Taking cells from a HLA-matched donor and inject into bone marrow into pt with refractory acute leukaemia
o In the 60s we thought this worked as we were removing diseases BM and replacing it with healthy BM
Kolb experiment here - basically white cells lead to CML remission
2
Q
Kolb et al (1990)
A
observed that in the pts with CML that relapsed after transplanting you could go back to the original donors and ask them to donate white cells. Infusing these white cells would turn a significant proportion of these patients back into remission
3
Q
Draw scFV and CAR T cell
A
4
Q
What is scFV?
A
- This is a fusion protein composed of the variable regions of heavy and light chains of monoclonal antibodies, connect via a serine-glycine linker sequence
5
Q
Structure of CAR T cell
A
CAR T cells consist of one scFv, linked via a spacer and transmembrane domain to intracellular molecules which elicit effector functions of the T cell which they are part of
6
Q
Eshhar et al 1993
A
- construction of first-gen CAR T cells – consist of intracellular domain of CD3z, responsible for ‘signal 1’ of T cell activation
7
Q
Maher et al 2002
A
- Following the construction and antigen stimulation of CD28-CD3z dual-signalling receptors, it was found that these were associated with increased IL-2 secretion as well as increased T cell proliferation with repeated antigen exposure in the absence of any exogenous stimulation, when compared with controls with receptors only expressing CD3z or only CD28
8
Q
What are other T cell costimulatory domains being investigated?
A
4-1BB (CD137), OX40 (CD134), DAP10 and ICOS
9
Q
Discuss manufacture of CAR T cells
A
- Manufacture ex vivo genetic manipulation of autologous T cells
- Do leukapheresis from a patient’s blood activate and modify the T cells to express a transgene encoding a tumour specific CAR, infuse CAR T back into patient after chemotherapy
- Gene transfer through retroviral or lentiviral vectors
10
Q
Sommermeyer et al 2016
A
- demonstrated in a preclinical model of Raji tumour-bearing immunodeficient mice receiving CAR T cells of distinct T cell subtypes. They found that there was synergistic antitumour activity of CD4+ and CD8+ T cell subtypes in a 1:1 ratio, compared to reduced antitumour activity when either CD8+ or CD4+ were omitted from the formulated product, shown by decreased survival and increased tumour growth seen via bioluminescence imaging
11
Q
Xu et al 2014
A
- analysis of 14 patients with B cell malignancies that had been infused with autologous CD19-redirected CAR T cells. They found that the expansion of these cells in vivo correlated with the frequency of TSCM cells infused in the first 6 weeks after infusion
- It is thought that naïve T cells (TN) differentiate to form TSCM and TCM cells that are capable of self-renewal, and that these provide populations of TEM and TEFF cells that are incapable of self-renewal, suggesting that the longevity of less differentiated cells provide a greater therapeutic efficacy
12
Q
Discuss mechanisms of CAR T killing/cytotoxicity
A
- CAR T cells are thought to facilitate their effects through the perforin and granzyme axis, the Fas and Fas ligand (FasL) axis, as well as the release of cytokines
- When EGTA is used to block perforin release, most CAR T cell-mediated killing is abrogated
- Has also been shown that ectopic Fas expression on tumour cells is able to improve CAR T cell activity
- To expand further, a secondary role of cytokines has been implicated in both types of therapy. This is thought to play a role in ‘bystander-lysis’, where cytokines that are released following activation of the redirected T cell cause lysis of antigen-negative tumour cells in close proximity to the antigen-positive tumour cells engaged, a process implicated in both BiTEs and CAR T cells
- Capable of serial killing, whereby one T cells is activated and kills several targets
13
Q
What is CD19 normally? Which malignancies is it involved in and on what proportion of these is it expressed?
A
- CD19 is a transmembrane glycoprotein required for normal B cell development
- It is expressed on 95% of haematological malignancies including B-ALL, chronic lymphocytic leukaemia and B cell non-Hodgkin’s lymphoma
14
Q
Describe B-ALL
A
- Most common in children and young adults and prior to such CAR T cell therapy, it had a median survival time of 6 months in adults, poor prognosis
15
Q
Levin 2014
A
- Previously classically tested on transplanted tumours - does not mimic TME
- Compared the tumour response of erbB2-specific CAR T cells in the Her2NG transgenic mouse – overexpresses human erbB-2 transgene under MMTV promotor – these mice progressively develop mammary tumours
- They found a single administration of CAR T cells lead to rejection of the primary tumour, but a few weeks later tumours relapses in mammary gland and remote sites. This could be controlled by repeated CAR T cell injections and one mice remained cancer free for 500 days following treatment
- They also found that CAR T cells could be used prophylactically a single injection of CAR T cells before the appearance of any mammary tumours could significantly delay the appearance of mammary tumours for several months
- Could we use them in high-risk patients prophylactically? – probably not (SEs)
16
Q
Dr Rosenberg’s group (2014)
A
The first group to report responses in human B cell lymphomas and leukaemias
Describe the outcome of phase I/II study in pts with advanced B cell malignancies using CD19-specific autologous CAR T cells resulted in 8/13 patients with complete remission
17
Q
Discuss Kymirah
A
- Kymriah was approved for patients aged 25 or younger with B cell acute lymphoblastic leukaemia (ALL), following success in clinical trials.
18
Q
ELARA trial
A
- The ELARA trial of 52 patients with follicular lymphoma showed an overall remission rate of 83% within three months of treatment with Kymirah. However, in this trial, 49% of patients developed cytokine release syndrome (CRS), an adverse side effect consisting of a systemic inflammatory response. This side effect has been responsible for several deaths in similar trials.
- For example, one study was terminated following death of 5 patients as a result of fulminant cerebral oedema.
19
Q
Describe Yescarta - what is it used for?
A
- Yescarta was the second CD19 CAR T cell therapy approved in 2017 by the FDA for adults with relapsed/refractory large B cell lymphoma.
20
Q
How are Kymirah and Yescarta administered?
A
- Both therapies are administered as a one-time infusion and require lymphodepleting preconditioning prior to infusion to create a favourable environment for CAR T cells, namely through the elimination of regulatory T cells
21
Q
How does CRS present? What is given to reduce it?
A
Fever
Fatigue
Anorexia
Hypotension
Tachycardia
anti-IL-6 receptor Ab or corticosteroids
22
Q
How does CAR T/BiTE neurotoxicity present?
A
- This neurotoxicity typically presents as confusion and delirium, although seizures and the aforementioned cerebral oedema may also occur
23
Q
Avery Posey October 2020
A
- showed, via multiple independent single-cell RNA sequencing databases and subsequent bioinformatic analysis, that mural cells, which have a critical role in blood-brain-barrier (BBB) integrity, express CD19!
- They confirmed this by performing IHC with an anti-human CD19 antibody in healthy subjects post-mortem; CD19 expression was found in cells adjacent to vessel basement membrane walls in perivascular area, along both smaller capillaries and larger vessels.
- They also found that the expression of CD19 in human cells is significantly higher than in murine mural cells, implicating possible limitations of mouse models to study immunotherapy-associated toxicity.
- Therefore, a cause of this neurotoxicity may be the targeting of CD19+ mural cells and subsequent BBB disruption.
- However, there are further points which this study might have expanded on; the group have not demonstrated that CAR T cell or BiTE targetting of CD19 in human mural cells is the direct cause of clinical neurotoxicity, and contribution of other processes, such as CRS, may also be important to the pathology.
24
Q
Roybal 2016
A
- constructed a T cell expressing a combinatorial circuit using a synthetic Notch (SynNotch) receptor, which, following target antigen binding, leads to cleavage of the receptor and release of a transcriptional activator.
- This inducible transcription factor (TF) is able to regulate transcription of a CAR recognising a second tumour antigen.
- Thus, this AND-gate circuit requires binding of antigen to both the SynNotch receptor and the CAR for subsequent T cell activation
- However, they still have little control over these cells after adoptive transfer
- A key observation in this study was their in vivo work; they used mice with a CD19 K562 tumour on one flank and a GFP+/CD19+ K562 tumour on the other.
- They injected T cells bearing an a-GFP synNotch regulating a CD19 CAR and monitored tumour growth.
- They found that in all animals, these cleared the dual-antigen GFP/CD19 tumours but did not clear the CD19+-only tumours which grew at similar rates to negative controls.
- This showed that these T cells would only activate and kill targets in response to multiple tumour antigens, a potential mechanism to spare ‘bystander’ cells expressing single antigens, limiting associated on-target toxicity, such as B cell aplasia or neurotoxicity.
- The study by Posey’s group builds upon this, identifying several genes, including CDJ4,* *HLA-DRA* and *LTP, which are highly enriched on B cells in comparison to brain mural cells which serve as potential targets for combinatorial recognition.
- A limitation of an approach like this may be that both antigens would have to be expressed at considerably high levels by tumour cells.
- Also found that the CAR stays on the surface of the cell for 8 hours – their model showed no cross-reactivity so maybe it is long enough
25
Q
Draw Roybals combinatorial antigens
A
26
Q
Describe NOT gates
A
- An example is where PD-1 and CTLA-4-based inhibitory CARs (where the antigen scFv is fused to the signalling domains of these inhibitory receptors) are able to recognise non-specific target proteins and attenuate T cell activation.
- Similarly, Posey’s group found genes more enriched in mural cells than B cells, such and BGN,* *FN1* and *SEMA5A, which could serve as future targets in the prevention of neurotoxicity via NOT gates
27
Q
Juillerat et al. (2017)
A
showed that the ability of these HIF-CAR T cells to kill target cells was significantly improved in hypoxic compared to normoxic conditions; although a caveat of this may be off-target effects in healthy hypoxic tissues, such as the bone marrow.
28
Q
Huang 2020
A
- Current challenge is predicting optimal number of cells to infusion – need better ctrl in space/time
- Off-switches permanently down-regulate activity of CAR T cells
- Developed on-switch CARs which can be activated by light in a confined space
- Cre-loxP expression of CD19-CAR induced by blue light via nuclear translocation and dimerisation of CRY2/CIB1 pair.
- Tested in Jurkat cell line (immortalised T cell line) and primary human T cells, subcutaneous injection into mice - saw long lasting spatial controlled action
- Address duration of light dosing regimens (24hrs in study) + how long CAR activity continued after light stimulus removal
- Blue light doesn’t penetrate tissue as well as red – further develop a system using red, or implantable LEDs
- Induction ability only lasted two days
29
Q
Mechanisms of resitance/relapse to CAR T
A
Loss of CD19 (2/3 of cases of relapse in ALL are due to this). Could generatee CAR with multiple antigens. CAR T cells taregtting CD20 and CD22 are in development. Another approach is to increase antigen expression on the target cells small molecule gamma secretase inhibitors have been shown to increase B cell maturation protein (BCMA) expression on the surface of myeloma cells by impairing the cleavage of surface expressed BCMA. The combination treatment with BCMA-targeted CAR T cells is now in clinical trial
CAR T transduction of the tumour. Give a tumour cell the CAR T. Masking. Only been described in a few cases.
Trogocytosis. CAR strips Ag off the tumour and allows them to evade. Mainly in preclinical models, not sure how relevant it is.
T cells dont work - they get overstimulated and exhausted - Equyem 2017 and Liu 2021
Evasion of appoptosis
Immunosuppressive TME- recruiing Tregs for example
30
Q
- Eyquem et al (2017)
A
- TRAC T cells
- Most current approach to making CAR T cells insert the CAR-encoding gene into the T cells without disrupting the resident TCR gene
- TRAC T cells – TCR alpha chain constant region
- Used a CRISPR-Cas9 gene editing approach.
- Authors introduced RNA encoding Cas9 into T cells as well as gRNA that targets the TRAC sequence – ds break in TRAC and a viral vectore with a CAR sequence flanked by sequences homologous to the TRAC sequence was introduced into the T cells – original TRAC sequence replaced by CAR sequences
- Deleted the endogenous TCR and replace it with a CAR TCR which was under the control of the promoter for the TCR. Expression regulated in the normal way of a TCR, avoiding overactivation of the T cell.
- They showed this avoided exhaustion in vivo. Used a B-ALL mouse model. Used FACs to determine the number of cells expressing exhaustion markers on day 17 following induction. Found conventional CAR T cells showed up to 50% positive expression for three markers of exhaustion by day 17 whereas less than 2% of the TRAC-CAR T cells did
- Also showed a marked improvement in survival in the mouse model
- Could the integration of viral vectors randomly in the genome be a cause of cancer?
- Idea for future exp compare how KOs of components of T cell exhaustion pathways compare with this CAR integration
31
Q
Liu et al (2021)
A
- generation of a switch receptor
- Generated a new chimeric antigen construct in which they linked the PD-1 EC domain to an IC activating domain of CD28 – instead of PD-1 transducing a negative signal, PD-1 transduces an activating signal
- Found that CD19-PD-1/CD28-CAR T cells had superior T-cell proliferation, cytokine production, and sequentially capability of killing PD-L1+ B-cell lymphoma cells in vitro and in vivo relative to the prototype, CD19-CAR T cells.
32
Q
Reinhard et al. (2020)
A
- designed a CAR targetting tight junction protein claudin-6 (CLDN6), a human tumour-specific antigen.
- They designed a nanoparticulate RNA vaccine, CLDN6-LPX, which promoted CLDN6 expression on the surface of dendritic cells.
- They used mice bearing CLDN6+ lung tumours and administered a subtherapeutic dose of mouse CLDN6 CAR T cells followed by CLDN6-LPX or control.
- It was found that, in the mice receiving CLDN6 CAR T cells and control, tumour growth was only delayed, but the mice receiving CLDN6-LPX demonstrated complete tumour regression and higher median survival.
- Therefore, RNA vaccines could be used clinically to promote CAR T cell expansion for treatment of solid tumours.
33
Q
Describe how ‘off-the-shelf’ CAR T cell therapy would work
A
- ‘Off the shelf therapy’ Take a healthy donor T cell population, ex vivo editing to KO the HLA which enables the T cell to be accepted in multiple populations without being rejected. You KO the TCR and then insert CAR T gene.
34
Q
Qasim, 2017
A
- Proof-of-concept evidence for allogeneic therapy
- 2 paediatric patients with B-ALL
- Used TALENs to engineer HLA-mismatched donor T cells
- Donor T cells were transduced with lentivirus to express CAR-CD19 and these were electroporated with TALENs to ablate CD52 and T cell receptor- alpha constant region
- CD52 so the cells could evade the conditioning therapy before transplantation and TCR-alpha constant region to minimise graft-vs-host disease
- Molecular remission seen within 1 month in both infants (1 had evidence of skin GVHD, managed with steroids)
- Saw long term remission
36
Q
Describe and draw structure of BiTEs and discuss their manufacture
A
- Recombinant soluble protein, consisting of two different ScFvs, usually one targeting CD3 on T cells and the other directed at tumour surface antigens, again, joined by a short flexible linker Fig 2
- Manufacture BiTEs can be secreted from mammalian cell lines and bacteria as recombinant single chain polypeptides in large quantities
- E.g. can be produced by Chinese hamster ovary cells that have integrate cDNA expression vector expressing BiTE protein
